The G12 subfamily of the heterotrimeric G proteins, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to mitogenesis and apoptosis. The three primary goals of our previous proposal were to investigate the specific signaling pathways leading to mitogenesis or apoptosis induced by the Ga subunits of G12 and G13 proteins, and to identify and characterize biochemically and functionally novel protein complexes formed by Ga12 and Ga13. We will extend our studies of the cellular and molecular mechanisms that are responsible for the mitogenic and apoptotic responses regulated by G12 subfamily. Our preliminary data indicated that Ga12 and Ga13 interact with and induce activation of the important signaling protein, serine/threonine protein phosphatase PP5. Notably, PP5 was shown to be critically involved in the regulation of mitogenic pathways by inhibiting the growth-suppressing signaling cascades and by activating growth-promoting signaling pathways. Additional important data showed that Ga12 and Ga13 interact with and induce activation of the serine/threonine protein kinase, LIM kinase 1, LIMK1. Importantly, LIMK1 is activated during mitosis and is involved in actin cytoskeleton reorganization. Therefore, this proposal will analyze novel molecular mechanisms that allow Ga12 and Ga13 to form specific signaling complexes with PP5 and LIMK1, thereby generating signals that both create either mitogenic or apoptotic responses in the cells. The detailed Specific Aims are: Aim 1. To address the role of the interaction of Ga12 and Ga13 with PP5 in signaling, mitogenesis, and apoptosis. (i) We will study the physiological role of Ga12/Ga13 with PP5 in signaling pathway in the regulation of the mitogenic or apoptotic responses. (ii) As PP5 interacts with heat shock protein 90, Hsp90 and Hsp90 is required for the Ga12-mediated proliferation response, we will study the role of Ga12 in PPf/Hsp90 function. (iii) As PP5 was shown to interact and inactivate apoptosis-signal-regulating kinase, ASK 1, and our data suggest that Ga12/Ga13 activate ASK1, thereby promoting apoptosis, we will study the role of Ga12/Ga13 in PP5/ASK1 function. Using fibroblasts and endothelial cells obtained from ASK 1 knock-out mice, we will address the physiological relevance of Ga12/Ga13-PP5/ASK1 pathway. Aim 2. To address the role of interaction of Ga12 and Ga13 with LIMK1 in signaling, mitogenesis and apoptosis. (i) We will characterize the mechanisms of the biochemical interactions of LIMK1 with Ga12 and Ga13. Using in vitro reconstitution assay, we will determine the role of protein interaction in Ga12 and Ga13-dependent activation of LIMK1 (ii) As LIMK1 regulates actin cytoskeleton and we have shown that LIMK1 is associated with the microtubule cytoskeleton in the cells, we will address the role of Ga12 /Ga13-induced LIMK1 in regulation of the actin and microtubule cytoskeleton. (iii) Using fibroblasts and endothelial cells obtained from LIMK1 knock-out mice, we will address the physiological relevance of the Ga12/Ga13-LIMK1 pathway. These studies are critical for understanding of the control of tissue proliferation and apoptosis, and to identify potential sites for therapeutic intervention.